1. Field of the Invention
The present invention relates to dyes that are suitable for staining ribonucleic acid polymers (RNA) and deoxyribonucleic acid polymers (DNA) and are particularly suitable for staining reticulocytes. The invention further relates to a fluorescent composition.
2. Discussion of the Art
In many cases, there is a need to detect RNA or substances containing RNA. For example, a reticulocyte is a substance known to contain RNA. Detection of reticulocytes in a blood sample and the enumeration of these reticulocytes are valuable to clinicians. The reticulocyte count of a blood sample is an indicator of erythropoietic activity, is an indicator of acute hemorrhage and hemolytic anemia, and is a measure of response to iron, vitamin B12, and folic acid therapy. As is known in the art, reticulocytes are precursors of mature red blood cells, and hence the term reticulocyte embraces the evolution and development of a mature red blood cell.
Detection and enumeration of reticulocytes in a blood sample have been carried out by both manual and automated methods by using appropriate stains such as new methylene blue (NMB), brilliant cresyl blue (BCB), acridine orange, and pyronin Y.
Vital staining with the dye new methylene blue is considered to be the reference method for reticulocyte determinations. In use, this dye precipitates RNA. The method is carried out manually and requires counting large numbers of cells with a microscope (for example, 500 to 1,000 cells). Consequently, the method is slow, tedious, and is subject to errors.
New methylene blue is nonfluorescent and true precipitated RNA is often difficult to differentiate from precipitated stain. New methylene blue stains by combining with intracellular RNA molecules to form a colored complex, which is visible under microscopic examination on account of its size and color. However, under certain conditions, the dye molecule itself can form complexes with other dye molecules. These dye/dye complexes are indistinguishable from dye/RNA complexes, with the possible result that counts are inaccurate and/or false positives for the specific cell type of interest are obtained. This problem is more likely when the dye solution has not been filtered to remove non-specific dye/dye complexes that have formed.
Acridine orange has been used for staining reticulocytes by both manual and automated procedures. Acridine orange, which is fluorescent, also precipitates RNA. Consequently, the use of this dye prevents quantitative estimates of RNA content because of potential quenching, a phenomenon caused by dye molecules interfering with one another in the energy transfer process. Under quenching conditions, the energy transfer process results in no net fluorescence emission.
Age profiles of cells based on RNA content being proportional to fluorescence are not reliable. Age profile is the key information sought to be derived in assays of blood cells. The function of the dye for staining reticulocytes is primarily to determine the percentage of immature red blood cells in the general circulation. The information is needed for determining the homeostasis of the blood cell formation, detection of blood cell related metabolic diseases, and the presence or absence of anemic diseases. Acridine orange has a great affinity for the plastic tubing in flow cytometers, thereby resulting in increased background, consequently requiring lengthy procedures for removing the dye from the flow cytometer tubing. In addition, cells stained by acridine orange are difficult to distinguish from the autofluorescent red cell peak. Finally, the reticulocyte count is usually lower than that obtained with new methylene blue. New methylene blue stains cells by combining with the intracellular RNA to form an insoluble precipitate within the cells. A discrete blue pattern is formed upon the interaction, thereby allowing for easy manual microscopic evaluation. Detection by means of acridine orange is performed on a flow cytometer. On account of the nature of the diffused pattern, it is difficult to differentiate the specific staining from acridine orange to that of non-specific background. Consequently, if the background is high, the net positive usually will be reduced and result in an artificially low value, compared with the more discrete new methylene blue staining method.
The use of pyronin Y requires prior fixation of the erythrocytes with formalin, is cumbersome, time consuming, and generally yields poor results. Moreover, pyronin Y has a very low quantum efficiency, leading to a very low fluorescent signals.
Accordingly, there is a need for providing a dye better suited for staining reticulocytes so as to provide a procedure for accurately determining reticulocytes in a blood sample.
A dye for staining reticulocytes preferably has the following properties:
1. The dye should not fluoresce in the absence of RNA.
2. The dye should have a good fluorescent quantum yield in the presence of RNA.
3. The dye must exhibit a certain level of water solubility and be able to penetrate the membrane of cells containing RNA.
4. The dye should preferably have an excitation peak at about 633 nm.
U.S. Pat. No. 4,957,870 involves the detection of reticulocytes, RNA, and DNA in human blood samples using a dye having the following structure:
wherein                X represents O, S, Se or C(CH3)2;        R1 represents an alkyl group having from 1 to 6 carbon atoms;        R2 represents an alkyl group having from 1 to 6 carbon atoms;        R3 represents fused benzene, alkyl group having from 1 to 6 carbon atoms, methoxy, or is absent;        R4 represents an alkyl group having from 1 to 6 carbon atoms, ethoxy, or is absent; and        n represents zero or an integer from 1 to 6.        